Microdialysis is a widely accepted technique in the study of brain neurophysiology. As a sample collection technique, it allows for the extraction of biogenic compounds which are produced in situ, from almost any brain area of conscious animals. The microdialysis probe can also be used to administer drugs directly into brain tissue. If two microdialysis probes are used, it is possible to administer a drug via one probe whilst monitoring distant neurochemical events via the other probe.
In studying mechanisms of drug action in vivo, the release of the following neurotransmitters, amongst other relevant biogenic compounds, is of general relevance: dopamine, gamma-amino butyric acid (GABA), noradrenalin, and serotonin. Also other compounds may influence neural activity, such as glutamate, glucose, taurine and choline. Furthermore, glycine may be a relevant neurotransmitter.
Following isolation, commonly using microdialysis, these compounds are subsequently quantitated by those techniques of analytical chemistry which provide the sensitivity and selectivity which are required to analyze small amounts of analytes in a complex mixture.
To date, the determination of pharmacologically relevant biogenic monoamines such as dopamine, noradrenalin, serotonin, and of pharmacologically relevant amino acids such as GABA and glutamate is mostly performed using HPLC in combination with either fluorescence or electrochemical detection. See, for example Clinckers et al. 2004; J. Neurochem. 89(4): 834-843 (electrochemical detection of norepinephrine, dopamine and serotonin) and Rea et al 2005; J. Neurochem 94(3); 672-79 (derivatization of GABA and Glutamate using o-phtaldialdehyde followed by HPLC separation and fluorimetric determination). The limited peak-to-peak separation, which is inherent to HPLC, only allows selective determination of said analytes to a certain extent.
WO2007028906 relates to a method for marking entities bearing a primary amine function essentially consisting in reacting in one step said primary amines with: a reagent (F) capable of forming a fluorescent compound with said primary amines, and a reagent (S) capable of substituting the reagent (F) with an ionizable group and forming a majority ion by fragmentation of said compound.
Recently, attempts have been made to analyze said pharmacologically relevant biogenic monoamines and amino acids in liquid chromatography—tandem mass spectrometry, or LC-MS-MS. This is an analytical technique which combines high selectivity with high sensitivity. Generally, in LC-MS-MS, a mixture of analytes is first separated on a column using liquid chromatography (LC). The one or more analytes which are eluting from the LC at a certain time are collected and transferred to the first MS. These processes commonly occur on-line. Herein, ionization of the one or more eluted analytes takes place, which ionization is commonly performed using Matrix Assisted Laser Desorption Ionization (MALDI) or Electron Spray Ionization (ESI) techniques. The corresponding one or more molecular ions thus formed are selected in the first MS. These molecular ions (which all have one m/z value but which may correspond to one or more different chemical identities) are then transferred into a collision chamber. In this chamber, the molecular ions disintegrate into characteristic fragments. One fragment which is characteristic for the analyte of interest, having one specific m/z value, is detected in the second MS.
LC-MS-MS can be employed to quantitatively determine the analyte in a sample. One way to allow quantitation is by comparative analysis of accurately known concentrations of analytes. The great selectivity of MS-MS which involves the selection of two characteristic ions (one for the molecular ions comprising the molecular ions of the analyte (parent ion), one for an ion fragment which is uniquely characteristic for the analyte (product ion)) not only helps to distinguish the analyte from the other compounds in the sample, it also greatly assists in the accuracy and the reproducibility of the quantitative analysis.
However, mass spectrometry is vulnerable to problems of interference. In this respect, one important factor that can affect the quantitative performance of a mass detector is ion suppression. Sample matrix, co-eluting compounds, and cross-talk can contribute to this effect. Recent experiments involving ESI of biological extracts have shown that the main cause of ion suppression is a change in the spray droplet solution properties caused by the presence of non-volatile or less volatile solutes. These nonvolatile materials (e.g., salts, ion-pairing agents, endogenous compounds, drugs/metabolites) change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector. The mass and charge of individual analytes are also factors in making a compound a candidate for ion suppression or in making one compound a source of ion suppression for another; cf. Annesley, T. M. Clinical Chemistry 2003 49:7, 1041-1044.
Fortunately, the presence of ion suppression or other deleterious effects can be evaluated via several experimental protocols. A particularly preferred protocol, which allows for the quantitative determination of a certain analyte of interest, even in the presence of highly variable ion suppression effects which may occur from batch-to-batch, involves the use of the corresponding isotopically labelled analyte as an internal standard.
Therefore, overall, LC-MS-MS is generally considered as a highly preferred analysis technique for the determination of small amounts of analytes in a complex sample.